Recent advances in plant cell and tissue culture have made possible the asexual production of multiple, genetically identical copies of a source plant, termed plant cloning. Such culture is typically begun with a unit of plant tissue containing totipotent plant cells obtained from a source plant. Totipotent cells have both the complete genetic information to develop into separate complete plants without involving the sexual union of gametes and the ready capacity to develop into complete plants if cultured in vitro under favorable conditions. Totipotent plant cells are obtainable from such areas of a plant as meristematic tissue and plant embryonic tissue. Meristematic cells are undifferentiated plant cells from which differentiated cells arise. Meristematic cells divide to yield other meristematic cells as well as differentiated cells that elongate and further specialize to form structural tissues and organs of the plant. Meristematic cells are located, for example, at the extreme tips of growing shoots or roots, in buds, and in the cambium layer of woody plants. Plant embryonic tissue can be found inside a seed of the source plant as a zygotic embryo (developed from a zygote, which is a cell resulting from the union of gametes during fertilization).
Plant production by tissue culture techniques has several advantages over production involving the sexual process of pollination and seed production. First, tissue culture is fast; plantlets can be obtained in much less time than required for flower production, pollination, consequent seed production and maturation, and germination. Second, tissue culture can be prolific; extremely large numbers of plantlets can be simultaneously produced. Third, plants produced by tissue culture are all genetically identical with predictable characteristics, except for an occasional spontaneous mutant. In contrast, each progeny plant resulting from sexual reproduction is the result of a genetic recombination process and so is genetically different from all other progeny plants. As a result, the characteristics of the progeny from sexual reproduction are not as predictable.
Because of the advantages of plant production by tissue culture, the process is being increasingly employed in such industries as ornamental plant production and agriculture. A current method of choice, because of its low cost, begins with the procurement of an explant or excised piece of totipotent plant tissue removed from a desirable source plant. The explant is placed on a culture medium (usually in the form of a gel) containing plant growth nutrients and plant growth hormones. Eventually, the explant evolves a macroscopically formless mass of tissue, frequently called callus tissue, (comprising undifferentiated or partially differentiated totipotent plant cells) which is transferred to an embryo-development medium containing hormones that stimulate the formation of somatic embryos. Somatic embryos appear similar to the zygotic embryos found in seeds, but, in contrast with zygotic embryos, are genetically identical to the source plant. As can be surmised, the above process of forming and culturing somatic embryos on gels or liquids requires aseptic techniques from start to finish.
Somatic embryos are too undeveloped to survive in a natural soil environment. Somatic embryos cannot yet produce their own carbon compounds or derive energy from photosynthesis and they lack their own energy source, such as an endosperm tissue. Therefore, somatic embryos are cultured with an energy source, such as sucrose. This culture medium is highly susceptible to invasion by microorganisms, which can result in death or retard the growth of the embryos Hence, the development of somatic embryos into viable plantlets capable of surviving outside aseptic culture conditions has heretofore proved to be a very difficult and inefficient process.
The ever increasing need for large numbers of genetically identical trees of optimal genotype in the various timber industries has prompted researchers to investigate tissue culture methods for production of tree embryos and plantlets. A classic paper by Hakman and von Arnold, J. Plant Physiol. 121:149-158 (1985) discloses the production of embryonic callus tissue from explanted zygotic embryos of Norway Spruce, Picea abies. Aseptic culture conditions are described which are suitable for generation of somatic embryos from the callus tissue. However, that paper notes poor success in producing viable plantlets from the somatic embryos.
Verhagen and Wann, Plant Cell, Tissue, and Organ Culture 16:103-111 (1989) discloses the initiation of embryonic callus formation and generation of somatic embryos from explanted Norway Spruce zygotic embryos. These researchers did not attempt to develop optimal conditions for producing plantlets from the somatic embryos.
Durzan and Gupta, Plant Science 52:229-235 (1987) discloses some success in producing somatic embryos of Douglas-fir, Pseudotsuqa menziesii, but poor success in converting the somatic embryos to viable plantlets.
As can be seen, while several researchers have developed techniques favorable to the production of somatic embryos of plants from explanted zygotic embryos or callus tissue, there is still a need for a practical method of converting cultured somatic embryos, or their equivalent, of plants to viable plantlets, especially plantlets that can survive outside aseptic culture conditions and grow into mature plants.